Indirect ELISA and Indirect Immunofluorescent Antibody Assay for ...

Exp. Anim. 59(1), 47–55, 2010

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Indirect ELISA and Indirect Immunofluorescent Antibody Assay for Detecting the Antibody against Murine Norovirus S7 in Mice Yota KITAGAWA1), Yukinobu TOHYA2), Fumio IKE3), Ayako KAJITA3), Sang-Jin PARK1), Yoshiyuki ISHII1), Shigeru KYUWA1), and Yasuhiro YOSHIKAWA1) 1)

Department of Biomedical Science, 2)Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, and 3)Experimental Animal Division, RIKEN BioResource Center, 3–1–1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan

Abstract: To evaluate murine norovirus (MNV) infection in laboratory mice, we attempted to develop an enzyme-linked immunosorbent assay (ELISA) system and an indirect immunofluorescent antibody (IFA) assay for detecting the anti-MNV-S7 antibody in mice. MNV-S7, which was isolated in Japan, was used in both assays. The antigen for ELISA was prepared by ultracentrifugation of culture supernatants of RAW 264 cells infected with MNV-S7. Positive sera were obtained from 6-week-old, female C57BL/6JJcl mice inoculated orally with MNV-S7. IFA against infected RAW 264 cells was able to discriminate positive sera from negative sera. Indirect ELISA was performed using 96-well ELISA plates coated with formalintreated MNV-S7 antigen. In this ELISA system, mouse sera obtained 2 weeks after infection or later showed significantly high OD values and were judged positive. An equal level of antiMNV-S7 antibody response was observed in BALB/cAJcl, C57BL/6JJcl, DBA/2JJcl, and Jcl:ICR mice; whereas, C3H/HeJJcl mice demonstrated slightly lower antibody production 4 weeks after infection. We also used this ELISA system to evaluate 77 murine serum samples obtained from 15 conventional mouse rooms in research facilities in Japan and found that approximately half of the serum samples contained antibody to MNV-S7. We found that some serum samples were negative for antibodies to mouse hepatitis virus and Mycoplasma pulmonis but positive for antibody to MNV-S7. The results suggest that the MNV infection is more prevalent than other infections such as mouse hepatitis virus and Mycoplasma pulmonis in conventional mouse colonies in Japan, as is the case in other areas of the world. Key words: enzyme-linked immunosorbent assay, immunofluorescent antibody assay, murine norovirus

Introduction The murine norovirus (MNV) was recently isolated

from immunodeficient mice that succumbed to a disease of unknown etiology [9, 23]. Genetic analysis of this agent has revealed that the pathogen (MNV-1) is close-

(Received 14 July 2009 / Accepted 16 September 2009) Address corresponding: S. Kyuwa, Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

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ly related to human norovirus, a member of the Caliciviridae family of positive-strand RNA viruses. In addition to MNV-1, several MNV strains have been isolated from laboratory mice [6, 10, 20]. Generally, MNV infection does not show any clinical symptoms in immunocompetent mice [23], except for occasional association with histopathological changes in the intestine and the spleen [14]. On the other hand, it sometimes induces noticeable symptoms such as diarrhea in immunodeficient mice, such as OTI/Rag1–/–IFN-g–/– and b2M–/– mice [20]. Persistent MNV infection has been reported in not only immunodeficient but also immunocompetent mice [4, 6, 14]. However, viral persistence is a viral straindependent phenomenon, since Karst et al. demonstrated that viral RNA was no longer present in visceral and mucosal tissues collected from immunocompetent mice 3 days after oral infection with MNV-1 [9]. Although MNV exhibits biological diversity as stated above, it has been found to comprise a single genogroup as well as a single serotype [20]. A recent study of a large number of mouse serum samples from research colonies in the United States and Canada identified MNV-1 reactive antibodies in 22.1% of serum samples [7]. In addition, the results of a serological survey that evaluated the contemporary prevalence of MNV in laboratory mice in Western Europe indicated that MNV is the most prevalent viral pathogen [13]. Although Goto et al. recently demonstrated MNV prevalence in Japan by RT-PCR [4], there is no information available on the MNV prevalence in laboratory mouse colonies in Japan based on serology. In this study, an indirect immunofluorescent antibody (IFA) assay and an ELISA system were developed using MNV-S7, a new MNV strain that was isolated in Japan. The results suggest that MNV infection is more prevalent than mouse hepatitis virus (MHV) and Mycoplasma pulmonis (M. pulmonis) infections in conventional mouse colonies in Japan, as is the case in other areas of the world [13, 15]. Materials and Methods Mice and sera Female, 6-week-old specific pathogen-free C57BL/6JJcl (B6), BALB/cAJcl, C3H/HeJJcl, DBA/2JJcl, and Jcl:ICR

mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). The breeding colonies except for Jcl:ICR were free of MNV when examined by RT-PCR (Dr. Goto, personal communication). Two experimental MNV-S7 infections of mice were carried out. The first one was a time course study using B6 mice. The second one was a strain difference study using five strains of mice. Mice were orally inoculated with 0.1 ml of 5 × 105 TCID50 of MNV-S7 on the day when they arrived at our laboratory. They were housed in isolation racks in a room maintained at a temperature of 23°C on a 12:12-h light-dark cycle, and were fed on commercial pellet CRF-1 (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum. After being anesthetized with isoflurane, blood was collected from naive and MNVS7-infected mice by cardiac puncture. Sera were collected and stored at –80°C until use. In the time course study, 4, 3, 3, 4, 4, and 2 B6 mice were sacrificed at 1, 2, 3, 4, 5, and 6 weeks after infection, respectively. Five naive B6 mice were used as negative controls. In the strain difference study, four each of B6, BALB/cAJcl, C3H/ HeJJcl, DBA/2JJcl, and Jcl:ICR mice were sacrificed at 4 weeks after infection. Five or four naive mice of each mouse strain were used as negative controls. The animal experiments were approved by the institutional animal care and use committee of the University of Tokyo and carried out in accordance with the guidelines for animal experimentation of the University of Tokyo, Japan. Sera from mice bred in conventional animal facilities were obtained from several universities and research institutions in Japan. Antibodies to MHV and M. pulmonis were examined by the use of MONILISA kits (Wakamoto Pharmaceutical, Tokyo, Japan) in accordance with the manufacturer’s instructions. Virus and cells MNV-S7, which was recently isolated from a conventional mouse in Japan (Genbank accession no. AB435515), was used. RAW 264, a macrophage-like tumor cell line was obtained from the RIKEN BioResource Center Cell Bank (Tsukuba, Japan) and cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 0.1 mM nonessential amino acids, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). The virus was

ELISA AND IFA FOR MNV-S7 ANTIBODY

propagated and titrated in RAW 264 cells using a conventional 50% tissue culture infectious dose (TCID50) assay. Briefly, viral suspensions were five-fold serially diluted (5–1 to 5–10) in 100 µl of DMEM medium to inoculate eight wells per dilution in 96-well plates, and a suspension of RAW 264 cells was added to each well (total volume of 200 µl/well). The cultures were maintained at 37°C, 5% CO2, and after 2 days, the cytopathic effect was manually recorded. TCID50/ml values were calculated according to the Reed and Muench method [16], where the tissue culture infectious dose is the virus dilution at which 50% of the wells contained infected cells. The results are presented as the –log mean of 4 replicates for each measured virus concentration. Indirect immunofluorescent antibody (IFA) assay RAW 264 cells were cultured in chamber slides (Nalge Nunc International, Rochester, New York) and inoculated with MNV-S7 at a multiplicity of infection (MOI) of 0.1. Eighteen to 20 h later, the infected cells were fixed in 4% paraformaldehyde and 8% sucrose in PBS for 20 min and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. The cells were treated with 5% bovine serum albumin (BSA) in PBS for 30 min at room temperature and incubated with diluted mouse serum in PBS containing 1% BSA for 1 h at room temperature. After being washed three times, the cells were incubated with Alexa Fluor 488-conjugated anti-mouse IgG (H and L chain)(Molecular Probes, Eugene, Oregon) diluted to 1:500 in PBS for 1 h at room temperature. The samples were then analyzed using a confocal laser-scanning microscope (LSM510 Version 2.02; Carl Zeiss, Jena, Germany). Preparation of MNV-S7 antigen for ELISA RAW 264 cells were inoculated with MNV-S7 at an MOI of 0.1 and then cultured in DMEM supplemented with 10% FCS in 150 mm dishes for 40–48 h. The culture supernatant was collected and centrifuged at 2,000 rpm (800 × g) for 10 min at 4°C to remove cellular debris. The resultant supernatant was collected and the viral titer was determined. One hundred and eighty milliliters of the supernatant that showed 105.83TCID50/0.1 ml were centrifuged at 25,000 rpm (105,000 × g) for 90 min in an SRP-28SA rotor (Hitachi Koki, Tokyo, Japan). The re-

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sultant pellets were resuspended in TN buffer (0.01 M Tris-HCl and 0.1 M NaCl, pH 8.0) and then layered on top of a 1-ml 30% sucrose cushion and centrifuged at 25,000 rpm for 90 min in the SRP-28SA rotor. The viral pellets were dissolved with 1.8 ml TN buffer containing 0.1% sodium dodecyl sulfate. After incubation at room temperature for 10 min, the suspension was centrifuged at 1,000 × g for 15 min at 4°C. The supernatant (106.61 TCID50/0.1 ml) was collected, divided into samples, and stored at –80°C until use. These procedures succeeded in achieving a concentration that was approximately 30 times higher than that estimated from the infectious viral titers. Purified virus was treated with 0.1% formalin for a week at room temperature, dialyzed against PBS at 4°C, and used as a viral antigen for ELISA. To examine the presence of infectious virus in the antigen, the antigen was filtrated with a 0.45-µm membrane filter and inoculated into RAW 264 cells, and the cytopathic effect was monitored. To further confirm the absence of infectious virus in the antigen, supernatant collected from RAW 264 cells that had been blindly passaged three times with the antigen was examined using the IFA assay. ELISA Aliquots (100 µl) of the MNV-S7 antigen diluted 1:100 in carbonate coating buffer (0.032 M Na2CO3, 0.068 M NaHCO3, pH 9.6) were added to each well of a 96-well plate (Thermo Fisher Scientific, Roskilde, Denmark) and allowed to incubate at 4°C overnight. After being washed three times with Tris-buffered saline (TBS) containing 2% Tween 20, the wells were incubated with carbonate coating buffer containing 5% skimmed milk (blocking buffer) at room temperature for 1 h. After being washed three times, 50 µl of mouse sera diluted with blocking buffer was added to each well and incubated at 37°C for 1 h. After being washed three times, horseradish peroxidase-conjugated rabbit antimouse IgG (g chain) antibody (ZYMED, San Francisco, California) diluted 1:2,000 in PBS was added to each well and incubated at 37°C for 1 h. After being washed four times, 100 µl of SureBlue™ TMB substrate (KPL, Gaithersburg, Maryland) was added and incubated at room temperature for 10 min. The reaction was stopped by adding 100 µl of 1 M H2SO4, and the OD450 was determined with a Wallac 1420 ARVOsx plate reader

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(PerkinElmer, Waltham, Massachusetts). To inhibit contamination by liquid waste from the ELISA system, we autoclaved all the liquid waste and the ELISA plates. Statistics Statistical analysis was performed using Student’s ttest and the chi-square test, with P